Many neurotropic viruses can invade the central nervous system (CNS), infect resident cells and establish acute or persistent infections. Flavivirus neuropathogenicity is defined by at least three major properties of the virus: (1) viremic capacity (the ability of a virus to produce and sustain the level of viremia sufficient for neuroinvasion), (2) neuroinvasiveness (the ability of a virus to enter the CNS), and (3) neurovirulence (the ability of a virus to replicate, spread, and induce neuropathology once within the CNS). Consequently, the research in our lab aims first to prevent the virus entry into the CNS and second to restrict its replication in the neurons. Mechanisms of flavivirus entry into the CNS have not yet been firmly established. To limit virus access into CNS, we have developed a set of the attenuated vaccine candidates using the strategies based on chimerization of a neurovirulent virus (JEV, WNV, SLEV or TBEV) with a non-neuroinvasive dengue type 4 flavivirus (DEN4) or naturally attenuated tick-borne Langat virus (LGT) and introduction of attenuating mutations. To restrict viral replication in the neurons, we have developed an effective strategy for selective control of virus neurotropism by targeting of viral genomes for cellular microRNAs (miRNAs) expressed in the brain. The most attenuated vaccine candidates were then evaluated for safety, immunogenicity, and their ability to protect mice and monkeys against challenge with wild-type virulent virus. In addition, a miRNA targeting approach was adapted to design environmentally-safe vaccine viruses restricted in their ability to infect and be transmitted by competent arthropod vectors (mosquitoes or ticks). Bi- and trivalent vaccines against JEV, SLEV and WNV diseases: Vaccine candidates were engineered by introducing multiple targets for the CNS-specific miRNAs into chimeric WN/DEN4, JE/DEN4 and SLE/DEN4 viral genomes in which the structural prM and E protein genes of the DEN4 virus have been replaced by those of wild-type virus: WNV, JEV and SLEV, respectively. In FY2019, we demonstrated that a single dose of bivalent vaccine formulations (admixture of miRNA-targeted viruses: WN/DEN4mirT + SLE/DEN4mirT; JE/DEN4mirT + WN/DEN4mirT or SLE/DEN4mirT + JEV/DEN4mirT) induced strong neutralizing antibody responses in immunocompetent C3H or immunocompromised B6 IFNRI-knockout mice and protected them against lethal challenge with parental wild-type viruses. In addition, mice immunized with trivalent vaccine (JE/DEN4mirT + WN/DEN4mirT + SLE/DEN4mirT viruses) were completely protected against wild-type JEV, WNV and SLEV virus challenge. Currently, the NFS is working to adjust the dose of each component in the different admixtures of combined trivalent vaccine to achieve the balanced antibody response against these three virulent wild type viruses. TBEV vaccines: The TBEV vaccine candidates are chimeric viruses carrying the prM and E structural protein genes of Far Eastern TBEV and remaining sequences derived from either (1) a non-neuroinvasive DEN4 (TBEV/DEN4), (2) a naturally attenuated, tick-borne LGT strain E5 (TBEV/E5) or (3) a most immunogenic LGT strain T1674 (TBEV/LGT). In FY2019 (Tsetsarkin et al., mBio 2019), we have improved live-attenuated TBEV vaccine candidates by combining multiple miRNA-targeting sequences for CNS-specific miRNAs in three distant regions of TBEV/LGT virus genome (i.e., in the duplicated capsid gene region, the duplicated E gene region, and the 3NCR) and achieved vaccine candidates that were avirulent and non-neuroinvasive in mice but induced strong protection against challenge with wild-type TBEV or unmodified TBEV/LGT in mice. Importantly, in nonhuman primates, a single dose of the TBEV/LGTmirT (T/1674-mirV2) virus induced TBEV-specific neutralizing antibody levels comparable to those seen with a three-dose regimen of an inactivated TBEV vaccine, currently available in Europe. Moreover, our vaccine candidate provided complete protection against a stringent challenge with a parental (not miRNA-targeted) chimeric TBEV/LGT in monkeys. Thus, this highly attenuated and immunogenic T/1674-mirV2 virus is a promising live-attenuated vaccine candidate against TBEV and warrants further preclinical evaluation of its neurovirulence in the CNS of nonhuman primates prior to entering clinical trials in humans. ZIKV pathogenesis: Recently, we generated a full-length infectious cDNA clone of ZIKV isolated during 2015 epidemic in Brazil (Tsetsarkin et al., mBio 2016) and used it as a convenient genetic platform for studies of virus-host interactions and vaccine development. We have established animal models (CD-1 and AG129 interferon receptor genes knockout mice) for studying the ZIKV infection and pathogenesis during vertical and sexual transmissions. In FY2019, we created a panel of viruses carrying target sequences complementary for the CNS-specific cellular miRNAs and for miRNAs enriched in male and female reproductive organ tissues. First, we traced dissemination of these viruses into the CNS and male reproductive tract of AG129 mice and found that ZIKV infection of the testis and CNS can be restricted independently by miRNAs selectively expressed in these organs (Tsetsarkin et al., Nature Communications 2018). In contrast, ZIKV infection of epididymis can only be blocked by co-targeting of the virus for testis- and epididymis-specific mir-141 and mir202 miRNAs. Our results support a model in which ZIKV infects the testis via a hematogenous route, while infection of the epididymis can occur via two routes: (1) hematogenous/lymphogenous and (2) excurrent testicular. These defined alterations in ZIKV organ tropism are an important step in the development of miRNA targeted live-attenuated ZIKV vaccine. In addition, we demonstrated that co-targeting of the ZIKV genome with brain-, testis-, and epididymis-specific miRNAs restricts virus infection of these organs, but does not affect virus-induced protective immunity in mice and monkeys against wild-type ZIKV. In the next step for studying the ZIKV infection in the female reproductive tract, we established an animal model and found that wild-type ZIKV infection of a pregnant AG129 female results in transfer of the virus from the heavily infected placenta to the fetus and induces microcephaly. We created a panel of ZIKV viruses expressing complementary targets for the placenta-enriched cellular miRNAs and demonstrated that some of them did not replicate in placenta and were not able to disseminate into fetal tissues of pregnant AG129 mice.